Table Of ContentBIOLOGICAL
O X I D A T I O N
BY
CARL OPPENHEIMER, M.D., Ph. D.
AND
KUR T G. STERN, Ph. D.
WITH THE COLLABORATION
OF W. ROMAN, Ph. D.
1939
Springer-Science+B usiness Media, B. V.
Softcover reprint ofthe hardcover lst edition 1939
ISBN 978-94-017-5835-2 ISBN 978-94-017-6291-5 (eBook)
DOI 10.1007/978-94-017-6291-5
PREFACE.
Two years ago, when I began to write the section dealing with desmolases of the
Supplement to the 5th edition of my book "Die Fermente und ihre Wirkun
gen", I was confronted with the necessity of integrating an hnmense material. It
became obvious that this would be impossible without the guidance of a theory capable
of encompassing the diverse facts and phenomena. In spite of the many attempts made
in that direction, no comprehensive theory was available. I was therefore compelled
to develop such a guiding theory. It was clear that any such theory would have to
reconcile the apparently contradictory views of W ARBURG and WIELAND. While this
would have been a very difficult task in 1926, when the last complete edition of
"Die Fermente" appeared, such a synthesis appeared feasible in 1937 owing to
the modifications which the theories of WARBURG and WIELAND had experienced in the
meantime (by these workers and their collaborators themselves) and owing to the con
tributions made by the pure theory (HABER, WILLSTATTER, MICHAELIS) as well as by the
study of specific enzyme reactions (THUNBERG, School of HOPKINS, KElLIN, v. EULER,
KUHN, SZENT-GYORGYI, etc.). I should like to emphasize that the theory of biological
oxidation as evolved on this foundation was and still is considered a tool made for the
task of arranging the experimental material in an orderly and logical fashion. The subse
quent period of writing the special parts of the "Supplement" has satisfied me that
thia aim has been attained. This unitary theory comprises the XVII. Main Part
of the "Supplement" to "Die Fermen te", whioh is now oomplete (The Hague,
Dr. W. JUNK). In this work I enjoyed the valuable colla.boration of Dr. W. ROMAN
(London) who assisted in the writing of several chapters of the General Part and also
wrote largely the section dealing with the desoriptive chemistry of the enzyme system.
Thus, Dr. ROMAN has also contributed to the present book.
My wish to take the general theory of biological oxidation out of the larger treatise
and to present it to the biochemists in the form of a monograph wa.s realized thanks
to the willingness of Dr. KURT G. STERN to cooperate in the undertaking. The present
book is the product of our collaboration.
CARL OPPENHEIKmR.
DII 1I4GUB.
PREFACE.
As an experimental worker in the field of biological oxidation I felt keenly the lack
of a comprehensive theoretical treatment of the subject. Therefore I welcomed the idea
of Professor C. OPPENHEIMER to develop the chapter on biological oxidation in his
Supplement to "Die Fermente" into a monograph. Given a considerable latitude
in the execution of the plan, I have endeavored to render the text less dependent on
other expositions. In doing so it was understood, however, that for fuller information
the reader should still be referred to the "main work" by OPPENHEIMER (1926), the Sup
plement, and to the original publications.
Besides making numerous alterations throughout the text, I have to assume res
ponsibility for the following sections which were either newly added or completely
rewritten: Redox Potentials in Heterogeneous Systems, Affinity and Rate of Reaction,
Semiquinones as Intermediate Steps of Oxidation-Reduction Systems, Photochemistry
of the Respiratory Ferment, Chemistry of the Hexnin Enzymes, Copper Proteins, Yellow
Enzymes, Carboxylase, Cozymase and other Pyridine Coenzymes, Protein Bearers of
Coenzymes, Quinoid Mesocatalysts, Fumaric Acid Catalysis and Citric Acid Cycle.
A large number of new references has been added to the bibliography which has been
completely rearranged. An attempt has been made to incorporate the more significant
contributions up to the end of 1938 when the manuscript was concluded. In order to
keep the volume of the book within reasonable limits, the detailed discussion of certain
aspects of cell respiration such as the effect of external factors and the mechanism of
the PASTEUR-MEYERHOF effect had to be omitted with regret. For a recent review of
the latter phenomenon reference is made to the comprehensive article by D. BURK (157b).
A number of colleagues and friends assisted generously in this undertaking. Dr.
DEAN BURK helped to disentangle the intricate questions of terminology and symbols
presented by the section on Energetics and Potentials. Dr. R. A. SHIPLEY read the
entire manuscript and suggested numerous changes pertaining to style and language.
Drs. W. ROMAN, K. SALOMON and J. L. MELNICK shared the task of proof reading. To
these and other colleagues who offered suggestions and criticisms, I am greatly indebted.
I also wish to thank my wife, Else, for her assistance in the arrangement of the
bibliography.
KURT G. STERN,
Laboratory of Physiological Chexnistry,
Yale University School of Medicine,
New Haven, Connecticut.
Biological Oxidation.
Table of Contents
Page
Prefaoe by C. OPPElNHElIMBR ........................................................ .
Prefaoe by K. G. STERN ........................................................... .
General Part
A. Introduotion................................................................. 1
B. Theories of oxido·reduction ................................................. 6
I. General ................................................................... 6
1. Historical............................................................... 6
2. General Theory of Oxido·Reduotion .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. . 7
a) Dehydrogenation and Hydrogenation as Electron Shifts. .. .. .. .. .. . . .. .. . 8
b) Energetios and Redox Potentials ...................................... 11
0) Catalysis ............................................................ IS
Oxidation by Oxygen, Aotivation of Oxygen .......... , ....... , .. .. .. .. . 14
Peroxide Formation ..................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Speoial Hypotheses on Oxygen Aotivation . . . . . . . .. . . . . . . . . .. . . .. . . . . .. . 18
Heavy Metal Catalysis ............................................... 19
Formation and Removal of Hydrogen Peroxide .... . . . . . . . . . . . . . . . . . . . . . 2lI
II. Extension and Reshaping of Previous Theories ............................... 'll1
1. Development of WIELAND'S Theory ....................................... 'll1
a) On the Nature of the Aotivation of Hydrogen ......................... 27
b) The Reile of Hydrogen Peroxide .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Model Experiments .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Hydrogen Peroxide in Biologioal Systems .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
0) Aooeptor Speoifioity .............................. , . . . . .. .. .. .. .. . . . . . 54
2. Development of WARBURG'S Theory .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
a) The Hemin Ferment of WARBURG ..................................... 40
b) The New Sohema of Cell Respiration ............................ , .. " . 41
0) The Yellow Enzyme and the Hydrogen Transferring Enzymes............ 49
d) The Pyridine Ferments ............................................... 48
III. Theory of Ohain Reactions ................................................. 44
Antioxidants and Inhibitors ........................................... 48
C. The Phenomena of Oxidative Catalysis ..................................... 50
I. Energetios and Potentials ................................................... 50
1) On the Energetios of Hydrogenation and Dehydrogenation. . . . . . . . . . . . . . . . . . 50
2. Tables of Redox Systems of Biological Interest.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3. Oxidation·reduction Potentials in Heterogeneous Systems ................... 59
II. Catalysis of Oxido·Reduotion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1. Heavy Metal Catalysis .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6i
6.
.) General Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Oxidative Oatalyses .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peroxidatic Effect of Heavy Metal Systems ............................ 70
Participation of Heavy Metals in Anaerobio Processes ................... 71
Inhibition of Metal Catalyais . .. . .. .. .. .. . .. .. .. .. .. . .. .. . .. . .. .. . .. .. . 711
VIII
Page
b) Hemin Catalyses ..................................................... 79
Action of Hemins in Model Systems ................................... 80
Catalyses by Hemoglobin and Related Compounds.. . . . . . . . . . . . . . . . . . . . . . 82
Heavy Metal-Containing Intermediary Catalysts (Mesocatalysts) .......... 84
2. Metal-Free Catalysis (Acceptor Catalysis) .................................. 85
a) General Considerations... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Autoxidizable and Non-Autoxidizable Systems .......................... 86
Enzymes and Intermediary Catalysts (Mesocatalysts) ...... , ....... , ., .. . 87
Effect of Metal Reagents on Acceptor Catalyses ........................ 88
Affinity and Rate of Reaction ........................................ 90
b) Model Systems and Intermediary Catalysts (Me&ocatalysts) .. .. . . .. . . .. .. . 93
General...... ....................................................... 93
Metabolite Systems ................................................... 93
Ascorbic Acid and Reductones ...................................... 93
Thiol Systems, Glutathione ......................................... 95
Quinone Catalyses ................................................... 99
Semiquinones as Intermediate Steps of Oxidation-Reduction Systems.... 100
Significance of Two-step Oxidation for the Oxido-Reductive Catalysis 104
Oxidative Deamination of Amino Acids by Quinones ................. 105
Acceptor Respiration ............................................... 108
III. Oxidative Catalysis via Peroxides ........................................... 118
1. Organic Peroxides ....................................................... 118
a) BAOH'S Oxygenases ................................................... 118
b) True Organic Peroxides ... , ......................... , ............. , '" 119
2. Heavy Metal Catalysis via Peroxides ..................................... 123
Special Part
D. The Enzyme System.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 127
1. Theoretical Fundament ..................................................... 127
1. The Enzymes of the Main Chain; Hydrokinases ........................... 127
2. Auxiliary Enzymes of Desmolysis .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. . .. 128
Auxiliary Enzymes of Terminal Desmolysis .... , ., ., ................... , ... 131
8. Survey of System of Desmolases ......................................... 131
II. Descriptive Chemistry of Enzyme System .................................... 133
1. The Hemin Systems ...... . . . . . • . . . • • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 134
a) General ............................................................. 134
Absorption Spectra .................. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 134
Theory of Photochemical Absorption Spectrum ......................... 136
Experimental Method ............................................. , ... 140
Tests of the Theory. ................................ , ............. , '" 141
Comparison of Light-Sensitivity of CO-Compounds .... , ............. , ... 141
Direct Spectroscopy of Hematin-Containing Enzymes ............... , .. ... 142
Position of Absorption Bands ......................................... 143
Reactions of Hemin Systems with Inhibitors ........................... 153
b) Constitution and Chemical Properties of Hemin Catalysts ...... , .. .. .. ... 156
Chemistry of the Cytochromes .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 158
Respiratory Ferment ........................... " ... , ., ............ '" 166
Catalase ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . .. 172
Peroxidase .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 178
Appendix: Copper Proteins (Polyphenol Oxidase).. . . . . . . . . . . . . . . . . . . . . . .. 181
2. Vitazymes.............................................................. 183
a) Flavinphosphoric Acid and Yellow Enzymes ........... , ............. , ... 184
Riboflavin .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 185
Riboflavin Phosphoric Acid and Flavin Adenine Dinucleotide ......•..... 189
The Yellow Oxidation Enzyme........................................ 190
Reversible Dissociation of Yellow Enzyme .......................... , ... 192
Link between Prosthetic Group and Bearer Protein. .. .•.. .. .. .•.. ....•.. 198
Absorption Spectrum ....................................••.....•..... 194
Oxidation-Reduction Potentials ............ , ......... , ... , . . .. .. . . .. •.. 195
Other Alloxazine Proteids .. . . . . . . . • . . . . . • . . . . • • . . . . . . . • . . . • . . . . . . . . . .. 196
IX
Page
b) Co-Carboxylase and Carboxylase ...................................... . 198
Chemistry of Vitamin Bl ............................................ . 198
Co-Carboxylase ...................................................... . 205
Carboxylase ........................................................ . 208
c) Ascorbic Acid .......................... , ... , ............. , ....... , .. . 208
Appendix: Reductones ................................................ . 210
S. Nucleotide Coenzymes and Enzymes ............ , ........... , ..... , ...... . 212
a) Pyridine Coenzymes ................................................. . 213
Diphosphopyridine Nucleotide (Cozymase) ., ., ., ....... , ....... , ., ... , ., . 213
Triphosphopyridine Nucleotide ......................... , ............. , . 220
b} Cophosphorylase .................................................... . 222
c) Protein Bearers of Nucleotide Coenzymes ............................. . 223
Apozymase ......................................................... . 224
Protein of Acetaldehyde Reducase ..................................... . 225
Diaphorase (Coenzyme Dehydrogenase) .... , ., ....... , ....... , ... , ... , .. . 226
Phosphorylase ...................................................... . 228
4. Quinoid Mesocatalysts. .................................................. . 228
a) Pyocyanine ......................................................... . 228
b) Chlororaphine. ....................................................... . 232
c) Toxoflavin .......................................................... . 233
d} Phthiocol and other Naphthoquinones ................................ . 237
e) Hallachrome ........................................................ . 238
E. General Biological Significance of Desmolysis ............................ . 241
1. General Picture of Desmolysis ........................................... . 241
2. The Stages of Breakdown .............................................. . 242
a) General ............................................................ . 242
b} Anoxybiontic Breakdown of Carbohydrates ............................ . 244
c) The Oxybiontic Terminal Desmolysis .................................. . 249
d) Amino Acids and Fatty Acids ........................................ . 254
F. Cell Respiration ............ " ., ., ... , ....... , .............................. . 258
I. Main Respiration and Accessory Respiration ................................. . 258
1. General Considerations ................. , ............. , .... " ....... , .... . 258
2. The Cyanide-Resistant Residual Respiratiun .............................. . 260
II. The Intermediary Catalysts (Mesocatalysts) .............. , .................... . 261
1. General ................................................................ . 261
Reactions with Molecular Oxygen ........................................ . 262
2. The Function of the Mesocatalysts ....................................... . 263
3. Place of Individual Mesocatalysts in Desmolysis ........................... . 265
4. Fumaric Acid Catalysis ................................................. . 268
Development of SZENT-GVORGVI'S Theory ................................ . 268
Applicability of the Theory to other Tissues and Donators ............. . 271
Arguments against SZENT-GVORGVI'S Theory .............................. . 272
5. Citric Acid Catalysis .................................................... . 274
KREBS' Theory ........................................................ . 274
Arguments against KREBS' Theory ........................ , ........... , .. . 275
Bibliography.................................................................. 277
Index........................................................................... 310
General Part *).
A. Introduction.
A comprehensive theory of biological oxidation catalysis is being developed on
the basis of two theories which only a few years ago were considered incompatible.
We refer to the theories of OTTO WARBURG and of HEINRICH WIELAND. The battle
cries "activation of hydrogen" and "activation of oxygen" have lost much of their
significance due to the progress made in our understanding of the underlying mecha
nisms. Strictly speaking, neither an activation of hydrogen nor of oxygen occurs
in biological oxidation. Both terms are eliminated in an e x act description of the
mechanism. However, they may be retained as a matter of convenience for the purpose
of describing certain important relationships.
If a mere description is intended it would be more correct to call the better known
biological oxidations dehydrogenation catalyses instead of oxidation catalyses.
In all of these processes - a few rather obscure instances excepted - a transfer of
hydrogen takes place. Theoretically it does not matter whether molecular oxygen
or another chemical compound functions as the acceptor of the hydrogen. This con
clusio~ is important because it adds the concept of theoretical uniformity of the
mechanism of such reactions to the recognition of the b i 0 log i c a I uniformity of
dehydrogena tion without oxygen (a n 0 x y b i 0 sis) and dehydrogenation with oxygen
(oxybiosis). Theoretically it is of secondary importance, whether the acceptor of
the hydrogen is autoxidizable, i.e. capable of further transfer of the hydrogen to
molecular oxygen as the acceptor; or whether it is not autoxidizable, i.e. capable of
hydrogen transfer to other chemical compounds; or whether the system may utilize
both types of acceptors. The point of primary importance is the hydrogen transfer
itself, representing the disturbance of an equilibrium whereby the hydrogen of an
organic compound, called the donator, is first labilized and then shifted to another
compound, the acceptor, in accordance with a thermodynamic potential. If the
acceptor is molecular oxygen, ultimate oxidation via hydrogen peroxide to water
takes place; if the acceptor is another chemical compound, anoxybiontic reactions
occur which we call oxido-reductions.
Processes of both kinds may occur spontaneously, i.e. without catalysis, if the
thermodynamic potential driving the general reaction
+ +
DR.J Acc ~ D Acc~ ............... (1)
is not opposed by kinetic hinderances. In case the reaction proceeds too slowly it
may be catalysed by inserting new reactants which speed up the process and which
thereby function as· catalysts. The general equation becomes:
*) The whole subject has been reviewed in detail by v. EULER (288) and by OPPENHEIMER (905)
Important special aspeots are treated in the publications (1169, 1171, 1373, 1374, 974, 105, 106
903, 904, 1302, 804). .
Oppenheimer-Stern, Biological Oxidation.
2 INTRODUOTION
+ + + + +
D~ Cat ~ D Cat H2 Ace ~ D Cat Acc~. . . . (2)
It follows that in each case the catalyst must represent a reversi ble oxidation
red u c t ion s y s t e m. It must be able to accept readily hydrogen from the donator in
accordance with the natural thermodynamic potential and to give it off just as readily
to an acceptor of higher potential. By virtue of this cyclic change from the oxidized
to the reduced state and back to the oxidized state the catalyst eliminates the inherent
kinetic obstacles in the system and promotes the completion of reaction (1) in accor
dance with the laws of thermodynamics.
Later it will be shown that there exist certain relationships between thermodyna
mics and kinetics. Here it may suffice to mention that a thermodynamic potential
which is too steep may inhibit the reaction and may require the interposition of
oxidation-reduction systems of intermediary potential range as catalysts. Certain
products of sugar decomposition, for instance, which possess a strongly negative
potential are unable to yield hydrogen directly to molecular oxygen or to the strongly
positive hemin systems. A whole series of catalysts with graded potentials is required
in this case to permit the hydrogen transfer from the sugar to oxygen via the hemin
systems.
The reaction scheme (2) represents the general mechanism of dehydrogenation
catalysis. In order to achieve complete oxidation it is necessary for the hydrogen to
be ultimately received by an autoxidizable redox system which in turn reduces oxygen
to hydrogen peroxide:
+ + + + +
DH2 Cat~D Cat H2 02~D Cat ~02.
Hydrogen peroxide is then transformed into water either by simple ("catalatic")
decomposition or by subsequent hydrogenation.
The greatest obstacle preventing a merging of the theories of W ARBURG and
WIELAND was WIELAND'S conception that dehydrogenation catalysts were specific
only with respect to the donator but not with regard to the acceptor (hydrogen peroxide
excepted (p. 34». In other words, once the hydrogen is "activated", it should go
to any acceptor including molecular oxygen. Subsequent experiments from the school
of WIELAND, notably those of BERTHO, have shown that the dehydrogenases of
WIELAND are not only "donator-specific" but also "acceptor-specific"; only such
catalysts which have an affinity for oxygen, and are therefore autoxidizable systems,
may effect terminal oxidation. The postulate of W ARBURG that only certain systems
can effect the ultimate oxidation is therefore justified. Today we know that the most
important autoxidizable catalyst of living cells is the respiratory ferment of W ARBURG.
The reconciliation of the two theories includes the polemics concerning certain
definitions. Strictly speaking there exists neither primary "activation" of hydrogen
nor of oxygen. If we express oxido-reduction in terms of electron changes, the "acti
vation" of the hydrogen consists in the labilization of hydrogen and subsequent libe
ration of an hydrogen ion (proton); the corresponding electron is taken up by the
acceptor which is thereby reduced. The "activation" of the oxygen, on the other hand,
consists in the charging of the oxygen molecule with two electrons which serve as
the points of attachment of the liberated hydrogen ions:
+ + + + + + +
DHz Oz~D 2 H+ 2E Oz~D -0-0- 2 H+~D HzOz*).
*) The symbol -0-0-is meant solely to indicate the charging of the oxygen; it does not imply
a suggestion as to the physical meaning of this type of charge.
